Mycobacteria are extremely important human pathogens. Their unusual cell wall structure and slow growth rate make them difficult to study from the standpoint of basic bacterial physiology. Mycobacteria have evolved into facultative intracellular parasites, capable of surviving with the phagocytic vacuole of the macrophage. It is likely that the ability of mycobacteria to acquire nutrients within the macrophage vacuole is tightly linked to intracellular survival and, therefore, to virulence. Knowledge of nutrient transport mechanisms for virulent mycobacteria will contribute directly to the design of novel therapeutic strategies and the development of new vaccines. The PI has isolated several mutants of Mycobacterium bovis BCG and M. tuberculosis which are deficient in transport of amino acids and peptides. In this competing continuation proposal, the PI will use classical and molecular genetic techniques and macrophage infection technology to address four specific aims: (1) to isolate and/or construct mutants of BCG and M. tuberculosis defective in transport and metabolism of arginine and oligopeptides; (2) to characterize transport of substrates by mutant and wild type BCG and M. tuberculosis; (3) to isolate and characterize transport regulatory mutants of BCG and M. tuberculosis; and (4) to examine the survival and growth characteristics, and nutrient transport activities, of mutant mycobacteria within mouse and human macrophages. These experiments will lead to a more complete understanding of the vacuolar environment which is the preferred ecological niche for mycobacteria within a mammalian host, and the nutrient uptake strategies employed by virulent mycobacteria to survive and grow in that intracellular environment.